This application addresses broad Challenge Area (06): Enabling Technologies and specific Challenge Topic, 06-DK-101: Development of cell-specific delivery systems for therapy and imaging. One of the major unsolved problems in drug delivery is how to transport poorly permeable molecules across membrane barriers and release them in specific cells or tissues. The way that Nature solves this problem is through membrane trafficking;cell impermeable ligands such as the iron-carrying transferrin protein bind cognate receptors, in this case the transferrin receptor, that reside in dynamic membrane trafficking pathways. The pathway occupied by the transferrin receptor involves rapid cycling between the cell surface and early endosomes, delivers transferrin to every cell of vertebrate animals, and represents a remarkable natural delivery vehicle. We previously demonstrated that derivatives of the synthetic membrane anchor N-alkyl-3[unreadable]- cholesterylamine exhibit a unique biological activity: when added to mammalian cells, they efficiently engage the membrane trafficking pathway occupied by the transferrin receptor, allowing them to rapidly cycle between the plasma membrane and early endosomes. In this way, N-alkyl-3[unreadable]-cholesterylamines can function as small artificial cell surface receptors;when linked to motifs that bind drugs or molecular probes, they can shuttle these compounds into cells and tissues via endocytosis. These unique biological activities result from functional mimicry of free cholesterol, a key component of plasma and endosomal membranes. By binding to the free cholesterol receptor NPC1L1, these compounds engage natural mechanisms that control cellular uptake and membrane trafficking of this critical membrane component. This biological activity enables N-alkyl- 3[unreadable]-cholesterylamines to dramatically enhance the volume of distribution (Vd) of linked peptides, drugs, and probes in vivo. Because transferrin is efficiently delivered to all cells in all tissues via the transferrin receptor, the ability of cholesterylamine-linked compounds to efficiently engage the same membrane trafficking pathway offers unprecedented potential to create transformative new tools for drug delivery. This proposal is focused the creation of low molecular weight cholesterylamine-linked peptides (<3000 daltons) that selectively deliver fluorescent molecular probes into specific tissues. The release of these probes in defined tissues will be mediated by tissue-specific endoproteases that cleave specific peptide substrates. To discover these peptide substrates, a "small-but-smart" library comprising 3375 fluorescent cholesterylamine- linked peptides arrayed as 512 pools of eight peptides each will be screened using confocal laser scanning microscopy after microinjection of the nematode C. elegans. The linked N-alkyl-3[unreadable]-cholesterylamine moiety will play two roles: it will enhance the Vd of the members of the peptide library, improving access to all tissues in vivo, and it will selectively display peptide substrates to the subset of proteases found on cell surfaces and in the early endosomal system. By flanking members of the peptide library with red and green fluorophores, fluorescence resonance energy transfer (FRET) will allow quantification of in vivo cleavage of members of the peptide library. Cleavage of these peptides by tissue-specific endoproteases will unload cargo in specific tissue types. Probe delivery into six easily identified tissues (neurons, muscle, hypodermis, gonad, intestine, execretory cell) of C. elegans will be quantified based on red, green, and FRET fluorescence images obtained from confocal microscopy. This information, combined with other pharmacokinetic parameters such as Vd, half- life in vivo, and toxicity, will be used to construct quantitative structure-property relationship (QSPR) models. These models are designed to guide the synthesis of non-toxic compounds with optimal tissue-targeting properties. To facilitate the translation of this approach to higher animal models, we will identify specific proteases that process cholesterylamine-linked peptides in C. elegans. Optimized peptide substrates will be converted into protease inhibitors by modification with C-terminal aldehyde and/or other protease inhibitory motifs at putative sites of scissile bonds. These inhibitors will be used to purify and identify proteases from extracts of C. elegans. Further studies using genetic mutants, RNAi methods, and expression of putative proteases in alternative tissues of C. elegans will be used to validate these target proteins. The proposed use of N-alkyl-3[unreadable]- cholesterylamines to transport molecular probes across membrane barriers, combined with the use of linked peptide libraries to identify substrates of tissue-specific proteases, represents a fundamentally new approach to tissue-specific drug delivery. PUBLIC HEALTH RELEVANCE: The research proposed here is highly relevant to the advancement of human health. Using small libraries of cholesterylamine-linked fluorescent peptides and high-content screening by confocal microscopy, we propose to identify relatively low molecular weight compounds that selectively release molecular probes in specific tissues of the model organism C. elegans. By identifying lead compounds from these studies, and identifying proteases that mediate tissue-specific release of linked probes, this research will lay the foundation for the development of novel methods for tissue-specific delivery of drugs and imaging agents in higher organisms.